A hippocampus-accumbens code guides goal-directed appetitive behavior

The dorsal hippocampus (dHPC) is a key brain region for the expression of spatial memories, such as navigating towards a learned reward location. The nucleus accumbens (NAc) is a prominent projection target of dHPC and implicated in value-based action selection. Yet, the contents of the dHPC→NAc information stream and their acute role in behavior remain largely unknown. Here, we found that optogenetic stimulation of the dHPC→NAc pathway while mice navigated towards a learned reward location was both necessary and sufficient for spatial memory-related appetitive behaviors. To understand the task-relevant coding properties of individual NAc-projecting hippocampal neurons (dHPC→NAc), we used in vivo dual-color two-photon imaging. In contrast to other dHPC neurons, the dHPC→NAc subpopulation contained more place cells, with enriched spatial tuning properties. This subpopulation also showed enhanced coding of non-spatial task-relevant behaviors such as deceleration and appetitive licking. A generalized linear model revealed enhanced conjunctive coding in dHPC→NAc neurons which improved the identification of the reward zone. We propose that dHPC routes specific reward-related spatial and behavioral state information to guide NAc action selection.


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Memories allow an organism to use past experience to op-24 timize current and future behaviors (1). The hippocampus 25 (HPC) is widely recognized as one of the main sites of 26 memory-related plasticity (2)(3)(4). Yet, while our understand-27 ing of memory processing within the HPC has greatly ad-  The HPC is a major part of the brain's limbic system, pro-37 cessing various kinds of memory by receiving highly pro- 38 cessed sensory information from the entorhinal cortex, and 39 sending behaviorally relevant outputs to diverse brain regions 40 via hippocampal Cornu Ammonis field 1 (CA1) and subicu-41 lum (5). Its elevated role for spatial memory is highlighted 42 by the presence of spatially tuned "place cells" that form a 43 "cognitive map" to support goal-directed navigation (6,7). 44 This cognitive map is further supported by neuronal coding 45 of navigationally relevant features such as borders, speed, 46 reward/goal locations (8)(9)(10)(11), but also non-spatial informa-47 tion such as that about future decisions or behavioral tasks 48 (12,13). The conjunction of this variety of encoded features 49 is suggested to provide a "scaffold" that supports the forma-50 tion of episodic memories (14). 51 Previous models of hippocampal memory processing as- 52 sumed largely homogeneous cell populations (15,16). How-53 ever, hippocampal principal neurons are increasingly rec-54 ognized as structurally and functionally diverse in terms 55 of their morphology, electrophysiology, transcriptomic cell 56 types, anatomical differences across its three axes, and pro-57 jection patterns (17, 18). Such heterogeneity may provide 58 the hippocampus with the intrinsic flexibility to meet the var- 59 ious demands of diverse environments (17), and to route task-60 relevant information to specific output targets (19, 20). 61 One target receiving strong projections from both dorsal and 62 ventral CA1 and subiculum is the nucleus accumbens (NAc) 63 (21, 22), a basal ganglia brain structure crucial for value- 64 based action selection (23, 24). Described as the "interface 65 between limbic and motor circuitry" (25), it has been sug- 66 gested as the main site of transformation from a hippocam-67 pal spatial code into a motivation-driven motor code (26). 68 In line with this, NAc neurons are spatially tuned, required 69 for spatial memory acquisition and consolidation, and dis-70 play task-dependent synchrony with dHPC neurons (27-30). 71 Indeed, disabling HPC→NAc projections diminished con-72 ditioned place preference (CPP) (31, 32), and optogenetic 73 stimulation was sufficient to artificially induce CPP (33, 34). 74 While these findings cement the NAc's role as an indispens-75 able hippocampus output node, surprisingly little is known 76 about which spatial, contextual and behavioral patterns of 77 hippocampal information the NAc receives. Here, we set out 78 to understand the specific contents of this information stream 79 during goal-directed navigation and its acute role in behav-80 ior. We hypothesized that the HPC may selectively route both 81 spatial as well as other task-relevant information to aid the 82 NAc in action selection. 83 To test this, we employed dual-color two-photon imaging, 84 capturing in vivo calcium signals of large populations of 85 neurons in dHPC, while using a projection-specific red flu-86 orophore to allow identification of NAc-projecting neurons 87 (dHPC →NAc ). Directly comparing dHPC →NAc activity with 88  Figure S1. (E) Schematic of dual-color projection neuron imaging method. Thy1-GCaMP6s mice were injected with AAVrg-Cre in the medial NAc and DIO-mCherry in dHPC. Representative coronal brain slice showing axonal mCherry expression in NAc (AP -1.3; left). Representative coronal brain slice stained with DAPI (blue) of dHPC, showing the outlines of the 3 mm cannula window used for imaging; scale bar represents 1 mm (second left). Field of view of one sample experiment showing Thy1-GCaMP6s expression in green and mCherry expression of putative NAc-projecting neurons in red; outlines show detected components used for analysis (right). (F) Two representative neurons' raw ("F"), denoised and deconvolved ("C") and event ("S") traces; red traces indicate mCherry co-expression (dHPC →NAc ). See also Figure S2. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01.
To understand the neural coding properties of large numbers 114 of dHPC →NAc neurons in behaving animals, we turned to 115 dual-color two-photon imaging in mice pan-neuronally ex-116 pressing the calcium indicator GCaMP6s, as well as the static 117 red marker mCherry in defined NAc-projecting neurons. For 118 this, we injected AAVrg-Cre in NAc and DIO-mCherry in 119 dHPC (CA1/subiculum border region) of Thy1-GCaMP6s 120 mice (37). This approach allowed us to obtain dynamic cal-121 cium signals both in a large majority of mCherry-negative 122 hippocampal neurons (dHPC -) and specifically mCherry co-123 expressing NAc-projecting neurons (dHPC →NAc ), simultane-124 ously within the same field of view using the same calcium 125 indicator ( Figure 1E and Figure S2). It allowed us to over-126 come constraints of electrophysiological studies such as rela-127 tively low sample sizes (19,20) or indirect connectivity mea-128 surements (38,39). Optical access to dorsal CA1 and pro-129 subiculum (also known as proximal subiculum (40, 41)) was 130 established by implanting a chronic hippocampal window af-131 ter virus injections ( Figure S1C) (42,43). Imaging data was 132 acquired after 5 days of behavioral training, was motion-133 corrected using NormCorre, and spatio-temporal components 134   Figure 2D). Within this population of place cells, we further 159 analyzed how specifically space is encoded, and found that 160 dHPC →NAc place cells had a higher spatial information rate 161 (47) (p < 0.001, Welch's t-test; Figure 2E). They also had 162 significantly higher levels of sparsity (p < 0.001, Welch's t-163 test; Figure 2F), a measure for how diffuse a neuron is firing 164 in the spatial domain (48). Furthermore, both the relative 165 calcium activity (activity inside the place field -activity out-166 side the place field; p = 0.0044, Welch's t-test) and reliability 167 of in-place field activity per lap (p = 0.014, Welch's t-test) 168 were significantly higher for dHPC →NAc place cells com-169 pared to dHPCneurons ( Figure 2G-H). These results sug-170 gest that the dHPC routes enhanced and more reliable spatial 171 information to NAc compared to the general dHPC popula-172 tion, in line with previous results pointing towards the neces-173 sity of dHPC →NAc projections for spatial memory expression 174 (31, 32).

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Place fields are modulated by local cue boundaries 176 and are overrepresented near the reward zone. Previ-177 ous studies found that place fields are often not homoge-178 nously distributed across the environment but can be modu-179 lated by salient environmental features such as textures, bor-180 ders, or reward/goal zones (11,49,50). We hypothesized 181 that information about such spatial features may be preferen-182 Example place fields with edges near belt texture boundaries. Primary place fields are indicated by color fill. Triangles mark start (no fill) and end (fill) points. Dashed black lines represent texture boundaries. (C and D) Place field edges accumulate near texture boundary areas. Histograms of dHPC -(green) and dHPC →NAc (red) neurons' place field start (C) and end (D). Dotted line and shade represent average and 95th CI of 1000x randomly shuffled place fields. Both dHPCand dHPC →NAc place field start and end positions are significantly overrepresented at the 99.9th percentile (dotted black lines) compared to a randomly shuffled distribution. Start (χ²(1, 5134) = 5.735) and end positions (χ²(1, 5217) = 4.397) of dHPC →NAc place fields are furthermore significantly overrepresented compared to the dHPCpopulation. (E and F) Place cells are overrepresented near reward zone in high success trials. (E) Histograms (bars) and kernel density estimations (KDEs; lines) of place field centers for dHPC -(left) and dHPC →NAc (right) neurons, split into high success trials (green/red) and low success trials (gray). Reward zone (Rew.; yellow) and anticipation zone (Ant.; bright yellow) are indicated as rectangles. (F) Proportion of place fields in reward and vicinity zone is significantly higher in high-success trials (colored bars) compared to low-success trials (gray bars) in NAc-projecting neurons (red) but not in dHPCneurons (green). 2-way ANOVA, F success (1,1) = 54.918, p < 0.001, F projection (1,1) = 0.958, p = 0.338, F interaction (1,1) = 2.969, p = 0.098. Post-hoc Welch's t-tests with Bonferroni correction: t dHPC -(3.561) = 3.698; t dHPC →NAc (3.479) = 8.671; tlow success (13.629) = 0.093, p = 1; thigh success (3.952) = 2.075, p = 0.215. Dashed line represents an even distribution of reward and anticipation zone. (G) A linear classifier shows significantly increased decoding accuracy of reward anticipation zone based on dHPC →NAc neural activity compared to that of sample size-matched dHPCneurons. Wilcoxon's t-test, W (9) = 5.0, n = 10 imaging sessions. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S4.

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Enhanced coding of low velocities in dHPC →NAc neu-240 rons. As correct performance in the spatial reward learning 241 task goes hand in hand with a reduction in velocity and an 242 increase of licking near the reward zone (see Figure S1K-243 N), we wondered if such non-spatial task-relevant behavioral 244 features were encoded by dHPC neurons and its projections 245 to NAc (9, 54,55). We hypothesized that NAc may have 246 privileged access to information on low velocities as mice 247 generally slow down near the reward zone, presumably to al-248 low for better discrimination and to engage in anticipatory 249 licking. In line with previous analyses of speed coding in 250 hippocampal and parahippocampal regions (8, 56), we av-251 eraged each neuron's calcium activity per velocity bin from 252 approaching the reward zone, mice also increasingly engaged 270 in licking behavior (see Figure 1B-D). Given the NAc's dual 271 role in appetitive and consummatory behaviors (57), we 272 wondered if licking behaviors might be reflected in the neural 273 activity of dHPC →NAc neurons. For this, we distinguished 274 between consummatory licking which occurs after a reward 275 is dispensed and allows the mouse to consume the reward 276 provided, and appetitive licking which is an operant behavior 277 that will lead to reward dispensation when performed at 278 the correct location on the belt. We found a significant 279 decrease of calcium activity during reward consumption in 280 both dHPCand dHPC →NAc populations ( Figure S5A-C).

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Appetitive licking, on the other hand, had no apparent ef-282 fect on neural activity in dHPCneurons but coincided with 283 a significant increase in calcium activity in the dHPC →NAc 284 population that began about one second before lick onset 285 and then fell below baseline 4 seconds after licking (Fig-286 ure 5A-C). We investigated if this population-averaged data 287  tracking, we monitored mouse orofacial movements using a 310 high-speed near-infrared camera ( Figure 6A-C). To test for a 311 causal role of excitatory dHPC →NAc projections in appetitive 312 behaviors, we injected animals with either CaMKIIa-driven 313 ChR2 or EYFP into dHPC and implanted light fibers in the 314 NAc (n = 4/3 mice; Figure 6D-F). After habituating mice to 315 run on the treadmill and receive rewards upon licking on the 316 lick spout, mice were given 5 mW of 473 nm 20 Hz (5 ms 317 duration) pulsed laser light for up to 10 seconds upon entry 318 into a hidden light stimulation zone. 319 We found that, shortly after stimulation onset, ChR2-320 expressing mice reliably showed increased mouth movement 321 for up to two seconds after stimulation, while we observed 322 no effects in mice expressing EYFP (p ChR2 = 0.0099, p EYFP = 323 0.617, paired t-tests; Figure 6G-H). In line with this, we also 324 found a significant deceleration of running on the treadmill 325 upon light delivery in ChR2 animals but not EYFP animals 326 (p ChR2 = 0.0381, p EYFP = 0.334, paired t-tests; Figure 6J-K). 327 These findings support the idea that dHPC →NAc projections 328 may not simply represent task-related non-spatial behavioral 329 features but may in fact represent a driving force in the gen-330 eration of task-relevant appetitive behavior. place cells (43 % vs. 31 %, p = 0.0027, χ²). Conversely, 349 place cells were significantly less likely to be speed-excited 350 than non-place cells (10 % vs. 15 %, p < 0.001, χ²), an ef-351 fect that was again more pronounced in dHPC →NAc neurons. 352 This shows that dHPC place cells, and in particular those pro-353 jecting to NAc, are more likely to be speed-inhibited, and less 354 likely to be speed-excited. 355 We next analyzed lick modulation of place cells and, sur-356 prisingly, found that the previously observed lick-related in-357 crease in calcium activity ( Figure 5B) was largely carried by 358 place cells and not by non-place cells ( Figure 7C). This ef-359 fect seems to be mostly carried by lick-excited neurons that 360 are significantly overrepresented in place cells compared to 361 non-place cells (8 % vs. 2 %, p < 0.001, χ²), particularly 362 in dHPC →NAc neurons (15 % vs. 3 %, p < 0.001, χ²; Fig-363 ure 7D). Lick-inhibited neurons, on the other hand, were 364 distributed equally between place and non-place dHPCand 365    One caveat of such conjunctive coding analyses is that in our 384 behavioral task, trained mice often show highly stereotypi-385 cal behavior, such that mice would mostly lick at one loca-386 tion where they would also slow down (see Figure 1C and 387 Figure S1K-N). In light of this, conjunctive coding could be 388 an epiphenomenon of collinear behavioral features. To ac-389 count for this collinearity, we modelled the influence of three 390 key behavioral features (space, velocity, and appetitive lick-391 ing) on the activity of each neuron by building a generalized 392 linear model for each neuron (GLM; Figure 8A). On aver-393 age, we found that our models could explain close to 40% 394 of the variance observed in our test datasets ( Figure S6A Figure S6C). We classified significant modula-409 tion as behavioral features whose shuffling led to a reduction 410 in variance explained in more than 95% of shuffled models. 411 We found that cells thus encoding space, velocity, and lick- trial-to-trial reliability and in-place-field activity. Our data 452 confirm previous suggestions that the NAc receives spatial in-453 formation from dHPC (26) and likely explains previously de-454 scribed spatial tuning of NAc neurons (29). The dHPC →NAc 455 population's observed enhanced spatial tuning and stronger 456 modulation of local cues parallels previously described dif-457 ferences between deep and superficial dHPC neurons (18). 458 Indeed, vHPC→NAc projection neurons have been shown 459 to accumulate mostly in deep layers (64), raising the possi-460 bility that previously observed anatomical-functional differ-461 ences could at least partly be explained by differences in pro-462 jection targets, thus adding another important layer of com-463 plexity to pyramidal cell heterogeneity (17).

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Place cells in CA1 and subiculum have long been appreci-465 ated to overrepresent reward/goal zones (53,65,66) in a way 466 that correlates with behavioral performance (35, 52). This 467 effect likely depends on the interaction of inputs from en-468 torhinal cortex layer 3, dopaminergic inputs from VTA, and 469 hippocampal NMDA receptors (36, 52, 67). While previ-470 ous studies suggested a role for a largely undefined dedicated 471 population of dHPC cells in this overrepresentation (65) and 472 differences have been observed along the radial dCA1 axis 473 (35), evidence for projection-specific reward zone represen-474 tations has remained sparse. Although synchronous activity 475 has been observed between dHPC and NAc during spatial re-476 ward learning tasks (38,68,69), in line with a central role of 477 NAc in reward learning (reviewed in ref (70)), our findings 478 present direct evidence of increased reward zone overrepre-479 sentation by dHPC →NAc neurons. Interestingly, a previous 480 study (19) showed projection-specific ambiguity of reward 481 zone activity depending on vCA1→NAc collaterals. Future 482 studies should investigate potential functional differences in 483 collaterals of dHPC projections (41).

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Successful behavioral performance in our spatial reward 485 learning task depended on a decrease in velocity and in-486 creased appetitive lick activity as mice approached the hidden 487 reward zone. In line with previous work (8,9,12,20), we 488 found a considerable proportion of speed-modulated dHPC 489 neurons. Interestingly, NAc-projecting neurons were more 490 likely to be speed-inhibited, suggesting an elevated role in 491 reward approach behaviors (see also ref (12)). Similarly, 492 we observed significant modulation of hippocampal neurons 493 during appetitive and consummatory licking (see also refs 494 (12,54)). This modulation was dichotomous: consumma-495 tory licking resulted in widespread suppression of dHPC ac-496 tivity, while appetitive licking led to enhanced activity in 497 dHPC →NAc neurons. Activity of dHPC neurons, and espe-498 cially those projecting to NAc, thus seems to mirror the activ-499 ity within NAc: Inhibition of NAc D1R medium spiny neu-500 rons (MSNs) has been observed during lick behavior (71)(72)(73) 501 and was shown to be both necessary (71, 74) and sufficient 502 (71, 75) for consummatory licking. We found that the origin 503 of these signals may be localized upstream, similar to recent 504 findings of an inhibitory permissive drive for licking in vHPC 505 projections to the NAc (72, 76) and that inhibiting these pro-506 jections facilitates licking (72). This suggests a similar in-507 volvement of dHPCand vHPC-accumbens projections for 508 consummatory licking.   The NAc has been described as a key node transforming mo-556 tivational information from the limbic system into motor be-   (89). Thus, our findings identify a direct role for the hip-566 pocampus in the generation of motor behaviors and build an 567 important bridge in our understanding of how sensory and 568 mnemonic processes guide behavioral action.

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Animals. Experiments were performed in adult male and fe-571 male mice. C57Bl/6 (n = 16) and Thy1-GCaMP6s (n = 6) 572 mice (GP4.3; The Jackson Laboratory, Bar Harbor, USA) 573 were bred under specific pathogen-free conditions. Heterozy-574 gous mice were group-housed with 12 hours reversed dark 575 light cycle at 21°C and ad libitum food/water access until 576 mice had recovered from surgery. Experiments were per-577 formed during the dark phase. All experiments were per-578 formed according to the Directive of the European Communi-579 ties Parliament and Council on the protection of animals used 580 for scientific purposes (2010/63/EU) and were approved by 581 the animal care committee of North Rhine-Westphalia, Ger-582 many.

Stereotactic virus injections. For stereotactic injection of 603
AAVs, mice were anesthetized with an intraperitoneal (i.p.) 604 injection of a mixture of ketamine (0.13 mg/g) and xylazine 605 (0.01 mg/g). Mice were head-fixed using a head holder 606 (MA-6N, Narishige, Tokyo, Japan), placed into a motorized 607 stereotactic frame (LuigsNeumann, Ratingen, Germany) and 608 warmed by a self-regulating heat pad (Fine Science Tools, 609 Heidelberg, Germany). After skin incision (5 mm) and re-610 moval of the periosteum, placement of the injection was de-611 termined in relation to bregma.   nal weight before the start of training. Habituation consisted 676 of progressive exposure of mice to manual handling by the 677 experimenter, obtaining milk rewards through a metal can-678 nula, gentle manual head fixation, the treadmill apparatus, 679 and, finally, head-fixed running on an unmarked treadmill 680 belt with random rewards provided through a metal cannula 681 lick spout after licking on it. The self-propelled treadmill 682 (Luigs & Neumann) consisted of three rotating cylinders cov-683 ered by a 7 cm wide and 360 cm long textile belt (Luigs & 684 Neumann) including six differently textured zones: horizon-685 tal and vertical glue stripes, glue dots, Velcro dots, vertical 686 tape stripes and upright nylon spikes. The reward zone for 687 imaging experiments was 30 cm long and was placed be-688 tween the end of the horizontal glue stripes and the begin-689 ning of the vertical tape stripes. The position of the mouse 690 was recorded via an optical sensor (Luigs & Neumann) mea-691 suring the rotation of the treadmill cylinder underneath the 692 mouse. Lick signals were measured by an analog piezo sen-693 sor; one full belt rotation was measured by an optical infrared 694 sensor. All signals were collected at 10 kHz by an I/O board 695 (USB-6212 BNC, National Instruments, Austin, USA) and 696 recorded using custom-written Python software. After two 697 weeks of habituation, mice were placed daily for 15 minutes 698 on a cued (see above) treadmill belt on which they needed to 699 lick on the metal spout in this hidden reward zone to obtain 700 a liquid reward in the form of condensed milk. Milk was 701 released by a miniature peristaltic pump (RP-Q1-S-P45A-702 DC3V, Takasago Fluidic Systems, Nagoya, Japan) that was 703 triggered by custom-written Python software via an I/O board 704 (USB-6212 BNC, National Instruments, Austin, USA). Af-705 ter five days of training, calcium activity was recorded while 706 mice performed the learned task.

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Infrared camera behavioral tracking. Headfixed mouse 708 behavior was continuously monitored by simultaneously us-709 ing two monochrome CCD cameras (Basler acA 780-75gm) 710 positioned at approximately 15 cm from the mouse. To cap-711 ture face dynamics, we used a high-resolution zoom lens (50 712 mm FL, Thorlabs MVL50TM23); for body dynamics, we 713 used a wide-angle lens (12 mm FL, Edmund Optics #33-714 303). Infrared illumination was provided via two 850 nm 715 LED arrays (Thorlabs LIU850A), and cameras were outfitted 716 with 850/40 nm bandpass filters (Thorlabs FB850-40). Both 717 cameras' positions were aligned for each mouse before the 718 start of recordings. Camera images were acquired at 25 or 719 75 Hz with 782×582 pixels using pylon Camera Software 720 Suite (Basler), each frame triggered by TTL pulses from the 721 recording software. Files were saved in compressed MP4 for-722 mat before further processing.  using the optical sensor detecting a full rotation of the tread-788 mill belt. Velocity was calculated using a Kalman filter ap-789 plied to the position signal. Behavioral data were then down-790 sampled to either match the sampling rate of camera track-791 ing (25/75 Hz) for training data or to match the sampling 792 rate of two-photon imaging (15/30 Hz), by using each time 793 window's arithmetic mean (velocity), median (position, lap 794 number, camera/scanner trigger), maximum (reward pump, 795 optogenetic trigger), or standard deviation (licking). Discrete 796 lick events were detected using Scipy's find_peaks function 797 with a minimum temporal distance of 0.33 s and a dynamic 798 minimum height threshold that was individually determined 799 by inspecting the synchronized infrared camera video. Lick 800 bouts were classified as lick events that were <2 seconds apart 801 from one another. Appetitive lick onsets were defined as lick 802 bout onsets that were preceded by at least 3 seconds absence 803 of lick events. Consummatory licking onset was defined as 804 the first lick event between 0.5 seconds up to 5 seconds after 805 reward pump trigger. Rewarded laps were defined as laps in 806 which the reward pump was triggered by the animal's lick-807 ing. Relative licking refers to the cumulative analog lick sig-808 nal per position bin or reward zone. Successful laps were 809 defined as laps in which mice showed at least 50 % of rela-810 tive licking in reward and anticipation zones and received a 811 reward. High success trials were defined as trials consisting 812 of at least 50 % of successful laps.

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Calcium signal processing. Two-photon imaging data was 814 processed using custom-written software in Python, largely 815 based on CaImAn (v1.6.2; (44)). Green and red chan-816 nel 16-bit TIFF stack files (512×512 or 1024×1024 pix-817 els times 9,000 or 18,000 frames) were first resampled to 818 1 px/µm before motion-correcting the red static channel us-819 ing NormCorre piecewise rigid (parameters: max_shifts = 820 40, num_frames_split = 2000, overlaps = 46, splits_els = 821 4, strides = 255). The motion-corrected red channel image 822 was then averaged over t and used for later identification 823 of mCherry-positive components. Motion correction vectors 824 were then applied on the dynamic green channel before us-825 ing constrained non-negative matrix factorization (CNMF) 826 for cell segmentation. Resulting traces were detrended and 827 deconvolved before filtering spatiotemporal components us-828 ing quality criteria followed by identity-and behavior-blind 829 manual curation based on visual inspection of spatial and 830 temporal footprints and the quality of deconvolution. To 831 identify mCherry-positive components, resulting spatial foot-832 prints were overlaid over the red channel average and a dy-833 namic threshold applied to visually match the optimal signal 834 discovery.

835
Place field analysis. Continuous belt positions were binned 836 into 45 bins of 8 cm length. For calcium signals, de-837 convolved events were used to avoid differential effects of 838 GCaMP6s calcium signal tails at different animal velocities 839 across space. Only time points with a velocity >2 cm/s were 840 considered for spatial tuning analysis. Spatial information 841 (SI) was calculated as follows (47): where i denotes the i-th spatial bin, o i is the animal's occu-843 pancy at spatial bin i, a i is the mean of deconvolved events at 844 spatial bin i, andā is the overall mean calcium activity. Place 845 cells were defined as cells whose SI was higher than the 95th 846 percentile of 1000x randomly position-shuffled SI values (see 847 ref (65) (48), using the same denotation as above: Place field reliability was calculated as the fraction of laps in calcium event activity at that position (see also ref (65)). The 892 two-dimensional centroid was calculated, and the resulting 893 angle transformed back to belt position to yield the place field 894 center. "Place fields near reward zone" refers to place cells 895 with COMs in either the anticipation zone (starting 30 cm 896 before reward zone) or reward zone.  Average velocity (K and L) and average licking (M and N) changes across belt position and days of mice tested with reward zone in "center" of belt (K and M) and "end" of belt (L and N). Blue dashed line represents presence of reward zone. n = 9 (cohort 1), n = 9 (cohort 2). All data are presented as mean ± SEM. *p < 0.05, ***p < 0.001.  Three representative fields of view (FOVs) from three different mice are shown, with singlelap spatial calcium activity of each 10 representative neurons, some putatively NAc-projecting. FOV information is shown on top, including numbers of identified dHPC -(green) and dHPC →NAc (red) neurons. FOVs shown are composites of CaImAn maximum local correlation images (caiman.summary_images .max_correlation_image) of GCaMP channel (green) and the averaged motioncorrected red channel with NAc-projecting mCherry fluorescence. Contours of ten representative neurons from each FOV are indicated with white outlines and numbers that refer to spatially averaged calcium activity below. Each neuron's normalized average calcium activity across 45 spatial bins per lap (y axis) is shown with key spatial information values above (SI: spatial information (47), Sp: Sparsity (48), Rel: reliability of each neuron's per-lap maximum activity to occur within the place field). Orange numbers refer to NAc-projecting neurons. Gray bars represent belt texture zones (60 cm). (C) Place field centers are not biased with respect to texture boundaries (percentile < 95th, permutation test) and the ratio is not different between neuronal populations (χ²(1, 5372) = 1.646, p = 0.1995). (D and E) Place field density between reward and anticipation zones compared to the rest of the belt for dHPC -(D) and dHPC →NAc (E) populations. (F) Reward and anticipation zone overrepresentation correlates with behavioral success (percentage of rewarded laps per session) for both dHPC -(green) and dHPC →NAc (red) populations (Pearson correlation; r dHPC -(15) = 0.622, p = 0.0107; r dHPC →NAc (10) = 0.619, p = 0.0421). ns: not significant, *p < 0.05.  between full and feature-shuffled models are significantly greater in dHPC →NAc neurons for position (Welch's t(523.23) = 5.984, p < 0.001), velocity (Welch's t(497.69) = 7.704, p < 0.001), but not for licking (Welch's t(552.63) = 0.3626, p = 0.717). All data are presented as mean ± SEM. ns: not significant, *p < 0.05, ***p < 0.001.